Abstract: Filoviruses, which include Marburg virus (MARV) and Ebola virus (EBOV), have a high fatality rate and still lack FDA approved therapeutics or vaccines for treatment. Filoviruses have a filamentous lipid- envelope and despite being discovered more than 40 years ago, not much is known on how they assemble and bud from the host cell plasma membrane during their replication cycle. Filoviruses encode seven genes including the viral matrix protein VP40, which regulates budding from the host cell plasma membrane. Understanding the budding processes of several viruses has had significant impact on elucidating the viral life cycle and identifying therapeutic targets. Indeed, VP40 has been suggested in some studies to be a viable drug target. The goal of this application is to examine the biochemical and biophysical effects of mutations found in VP40 in patients from previous outbreaks. In specific aim 1, we hypothesize that an anionic to neutral amino acid substitution on the purported interfacial-binding surface of MARV VP40, permits enhanced lipid-binding properties and a reduction in the anionic charge density of lipid membrane required for in vitro lipid binding and cellular assembly at the plasma membrane. We will use in vitro biophysical assays, quantitative live cell imaging, and VLP release assays to come to quantitative and confident conclusions on the role of this mutation in MARV VP40 assembly and budding. Specific aim 2 will investigate the effects of a EBOV VP40 mutation, discovered in patients during the course of the recent EBOV outbreak, which lies in the purported membrane-binding interface of EBOV VP40. Lipid-binding, cellular assembly and VP40 oligomerization properties will be investigated. Quantitative assays of VP40 oligomerization state, VP40 lipid binding, and budding of VLPs will be assessed to decipher the role of this VP40 C-terminal domain mutation on EBOV VP40 biophysical and biochemical properties. Taken together, these studies should produce new and important mechanistic insight into biochemical consequences of VP40 mutations that occur during passage of virus through humans.